Genomics and, more recently, proteomics, have become major industrial activities, often associated with high density, microarray device formats which enable numerous parallel assays.
There has been significantly less activity, however, relating to microassay formats for testing or screening biological cells, e.g. where the functionality of an expressed protein or the activity of a new drug can be studied. A potential advantage of such formats, were they to be more universally adopted, could be a reduction in the high attrition rates in the discovery of pharmaceutical compounds during pre-clinical or clinical trials.
However, conventional cell-based assay devices are relatively limited in terms of the types (or functionalities) of tests that can be performed on cells.
For example, Aurora Biosciences™ have developed a high-throughput GPCR assay in a 3456-well microplate using a human T-cell line (Hong Xing, Hung-Cuong Tran, Thomas E. Knapp, Paul A. Negulescu, and Brian A. Pollok, A Fluorescent Reporter Assay for the Detection of Ligands Acting Through G1 Protein-Coupled Receptors, Journal of Receptor and Signal Transduction Research, Vol. 20(No. 4), (2000), 189-210).
Molecular Devices™ have developed FLIPR™ assays which can be used to measure the changes in intracellular calcium ion concentration and membrane potential from cells contained in either 96- or 384-well plates.
WO 01/25769 describes a substrate for determining and/or monitoring electrophysiological properties of ion channels. The substrate makes use of the so-called “patch-clamp” or “voltage-clamp” technique in which a seal is formed at a portion of an ion-channel-containing cell membrane in order to electrically isolate a measuring electrode at the seal from a reference electrode. The measuring electrode can then measure the electrical potential at the sealed-off portion of the cell membrane. This enables the study of e.g. ion channel responses to drugs.
The substrate described in WO 01/25769 has a plurality of sites, each of which has a measuring electrode associated therewith, and one or more reference electrodes. The electrodes described act solely as sensors, for electrochemical (impedance) measurements. The sites are in the form of wells having piping which applies suction to cells placed in the wells, thereby positioning the cells in the wells and forming seals to the cell membranes.
There has however been relatively little development of cellular assay techniques for studying electrically active cells such as myocytes, mast cells or neurons. Such cells are closely associated with major disease states including infarction and neurological disorders like Parkinson's disease. For example, WO 98/54294 describes an apparatus in which an array of microelectrodes are disposed in a cell culture chamber so that detection and monitoring of a voltage signal applied across each electrode can provide information on the electrical characteristics of individual cells. The geometry of electrodes in WO 98/54294 is designed such that they evoke an action potential through an injection of current, although there must be a good electrical contact between the cell and the electrode surface. In addition, impedance measurements made in WO 98/54294 require there to be an electrical contact between the cell and the sensing electrodes.
WO 02/08748 proposes a further substrate for manipulating membrane potentials of living cells via electrical stimulation. This substrate is based on a multiwell plate. An array of electrodes is dipped into the wells, so that an electrode pair is introduced into each well. An aim is to electrically stimulate cells held in the wells while performing optical analysis of transmembrane potential changes.
A drawback, however, of the substrate disclosed in WO 02/08748 is that the electrical fields needed to induce cell stimulation can lead to electrolysis of the well solution. Electrolysis results in bubble formation and a consequent change in chemistry (e.g. pH) of the solution. This in turn can affect the electrical response and indeed the viability of the cells (e.g. due to electroporation).
To limit the extent of electrolysis, WO 02/08748 proposes stimulation protocols which are only up to about one minute in duration. Also the electrical pulses which form the stimulation protocols are kept short, and polarities are inverted to balance charge.